Fig 1.
Istradefylline promotes Aβ generation.
(A) HEK293/APPswe cells were treated with Carbidopa, Istradefylline, Amantadine or Beserazide at indicated concentrations for 2 h followed by the detection of total Aβ levels in the culture media. The chemical structure of each compound is presented. (B) The secreted total Aβ levels and cell viability in response to Istradefylline at a range of concentrations. (C) The endogenous Aβ production of SH-SY5Y cells upon the treatment with 0.1% of DMSO (Vehicle), Istradefylline (Istrad.), Preladenant (Prelad.) or Tozadenant (Tozad.) at 30 nM for 24 h was examined. (D) The primary neuronal culture of APP/PS1 mouse was stimulated with vehicle (0) or Istradefylline at indicated concentrations for 24 h followed by the measurement of Aβ. T-test was used here. (E) The level of Aβ42 in the cortex of APP/PS1 transgenic mouse after chronic treatment with vehicle or Istradefylline. N = 11/group. (F) The level of Aβ40 in the cortex of APP/PS1 transgenic mouse after chronic treatment with vehicle or Istradefylline. N = 11/group. Data are mean +/± SEM of at least three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Fig 2.
Istradefylline promotes Aβ generation and γ-secretase activity through targeting A2ARs.
(A) A2AR antagonists increase the total Aβ level. HEK293/APPswe cells were treated with Istradefylline (Istrad.), ZM 241385 (ZM) or SCH 442416 (SCH) at indicated concentrations for 2 h and the total Aβ productions were then determined. The chemical structure of each compound is shown. (B) Caffeine-modulated total Aβ generation in HEK293/APPswe cells. Cells were treated with caffeine at 1 or 10 μM for 2 h followed by the determination of total Aβ levels in the culture media. (C) Quantification of A2AR mRNA level after transfection with scrambled or A2AR siRNA in HEK293/APPswe. (D) The cAMP response of the cells to agonist treatment. HEK293/APPswe cells were co-transfected with pGloSensorTM-22F cAMP plasmid and scrambled or A2AR siRNA followed by the stimulation with 1% of DMSO (Ctrl) or 30 nM of CGS 21680 HCl (CGS). Data are shown in arbitrary luminescence units. (E) The knockdown of A2AR reduces antagonists-increased Aβ generation. Cells transfected with scrambled or A2AR siRNA were treated with 0.1% of DMSO (Vehicle), Istradefylline (Istrad.), ZM 241385 (ZM) or SCH 442416 (SCH) at 30 nM followed by the determination of total Aβ. (F) The knockdown of A2AR reduces Istradefylline-increased γ-secretase activity. Cells transfected with scrambled or A2AR siRNA were stimulated with vehicle or Istradefylline (Istrad.) at 30 nM followed by the measurement of γ-secretase activity. Data are mean + SEM of at least three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 vs. control within the group. ##, p < 0.01; ###, p < 0.001 vs. control of scambled.
Fig 3.
The increased Aβ generation induced by Istradefylline is not through PKA, PKC or β-arrestins.
(A) A2AR ligands-modulated cAMP response in HEK293/APPswe cells. Cells were transfected with pGloSensorTM-22F cAMP plasmid and then stimulated with CGS 21680 HCl (CGS), ZM 241385 (ZM), Istradefylline (Istrad.) or SCH 442416 (SCH) at indicated concentrations. (B) A2AR antagonists block agonist-induced cAMP increase. Cells were pre-treated with ZM 241385 (ZM, 1 nM), Istradefylline (Istrad., 100 nM) or SCH 442416 (SCH, 1 nM) followed by the stimulation with CGS 21680 HCl (CGS, 1 μM). (C) The effect of A2AR ligands on Aβ production in the absence or presence of H89. Cells were incubated without or with 10 μM of H89, prior to the treatment with 0.1% of DMSO (Ctrl), CGS 21680 HCl (CGS, left) or Istradefylline (Istrad., right). H89 was present in the indicated groups throughout the experiments. (D) The effect of PKC activator or Istradefylline on Aβ production in the absence or presence of GO6983. HEK293/APPswe cells were pre-treated without or with 1 μM of PKC inhibitor GO6983 followed by the treatment with 0.1% of DMSO (Ctrl), 1 μM of PMA (left) or 30 nM of Istradefylline (Istrad., right). (E-G) The effect of β-arrestin1 or 2 on Istradefylline-modulated increase of Aβ generation. (E, F) Quantification of mRNA levels after transfection with scrambled, β-arrestin1 (E) or β-arrestin2 (F) siRNA in HEK293/APPswe cells. (G) After the transfection with scrambled, β-arrestin1 or β-arrestin2 siRNA, HEK293/APPswe cells were subjected to the treatment with 0.1% of DMSO (Ctrl) or Istradefylline (30 nM) followed by the detection of secreted total Aβ levels. (H) Representative images and the accompanied analysis of A2AR endocytosis upon the treatment with 0.1% of DMSO (Ctrl), CGS 21680 HCl (CGS) or Istradefylline (Istrad.) at 1 μM in HEK293 cells transfected with Flag-A2AR. Scale bar = 10 μm. N = 18–24 cells. Data are representative or mean +/± SEM of at least three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001. PMA, phorbol 12-myristate 13-acetate.
Fig 4.
A2AR colocalizes with γ-secretase complex in endosomes and interacts with PS1.
(A-E) Representative images showing the subcellular distribution of PS1 and A2AR with endocytic markers. HEK293 cells were co-transfected with indicated protein constructs and pMLink-Pen2-NCT-APH1aL and stained for EEA1 or Flag-PS1 or labeled with lysostracker. (A) Subcellular colocalization of CFP-PS1 (C-P), A2AR-YFP (A-Y) and EEA1. (B-D) Subcellular colocalization of Flag-PS1 (F-P), A2AR-CFP (A-C) and Rab5-GFP (Rab5-G, B), Rab7-GFP (Rab7-G, C), Rab11-GFP (Rab11-G, D). (E) Subcellular colocalization of CFP-PS1 (C-P), A2AR-YFP (A-Y) and lysotracker (lyso). Scale bar = 10 μm. White box indicates the enlarged area. N ≥ 10 cells for each group. The number on the right-top corner presents the Mander’s colocalization coefficiency of two channels. (F) Representative image showing co-IP of γ-secretase complex components or BACE1 with Flag-A2AR in HEK293T cells. Cells, transfected with four γ-secretase complex components (PS1, NCT, APH1aL and Pen2) or BACE1 and β-gal or Flag-A2AR, were lysed in the buffer containing 1% of CHAPSO or 1% of TritonX-100 (TX-100) and immunoprecipitated with anti-Flag slurry. The antibodies were previously verified [11]. (G) Flag-A2AR co-immunoprecipitates with HA-C99, but not HA-APPswe. Cells were transfected with HA-APPswe or HA-C99 without or with Flag-A2AR and then lysed, immunoprecipitated and blotted. Representative image is shown. (H) A2AR interacts with PS1 in APP/PS1 mouse brain. Brain membrane fractions were extracted from a 9 month APP/PS1 mouse. PS1 was immunoprecipitated with anti-PS1 antibody (1563 as labeled in the figure) and samples were subjected to Western-blot analysis. The antibody recognizing the endogenous A2AR has been verified [58]. (I) FRET efficiency of A2AR-CFP with YFP-fused other proteins. HEK293 cells were transfected with A2AR-CFP and YFP alone (Y), A2AR-YFP (A2AR-Y), APH1aL-YFP (APH1aL-Y), NCT-YFP (NCT-Y), YFP-PS1 (Y-PS1) or BACE1-YFP (BACE1-Y). Cells were then fixed and subjected to acceptor photobleaching FRET experiment. N ≥ 30 cells per condition. Data are mean + SEM. *, p < 0.05; ***, p < 0.001.
Fig 5.
Istradefylline, but not CGS 21680 HCl, attenuates the interaction between A2AR and PS1 and influences the internal conformation of PS1.
(A, B) HEK293 cells co-transfected with CFP-PS1 and A2AR-YFP were treated with Istradefylline (30 nM) for 0–60 min followed by the determination of FRET efficiency using acceptor photobleaching FRET (A) or FRET SE technique (B). Both the FRET efficiency and the attribute units of YFP or CFP intensity are presented in B. N ≥ 20 cells per condition. a.u., arbitrary units. Alternatively, the transfected cells were treated with Istradefylline (C) or CGS21680 HCl (D) at indicated concentrations for 30 min followed by acceptor photobleaching FRET assay. (E) Representative image showing Istradefylline reduces the co-IP of PS1 with Flag-A2AR. HEK293T cells were transfected with Flag-A2AR alone, PS1 alone or co-transfected with Flag-A2AR and PS1. Cells were treated without or with 30 nM of Istradefylline and then lysed with 1% of TritonX-100 IP buffer, followed by immunoprecipitation and western blotting. (F) PS1 conformation revealed by the internal FRET efficiency of CFP-PS1-YFP. HEK293 cells were transfected with CFP-PS1-YFP (C-PS1-Y) followed by the treatment without or with 30 nM of Istradefylline. As a control, cells were co-transfected with CFP-PS1-YFP (C-PS1-Y) and pMLink-Pen2-NCT-APH1aL (PNA). N ≥ 30 cells per condition for FRET assays. Data are mean +/± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Fig 6.
Scheme of A2AR modulates γ-secretase activity and Aβ generation.
(A) Reported scheme of A2AR agonist promoting Aβ production. Agonist binding to the receptor activates Gs protein leading to the activation of cAMP/PKA signal pathway. The elevated cAMP promotes APP expression and γ-secretase activity resulting into increased Aβ generation. (B) A scheme of A2AR antagonist-modulating Aβ generation revealed by our study. A2AR forms a complex with γ-secretase complex composed of PS1 (blue), APH1 (red), NCT (orange) and Pen2 (dark blue), and binds with PS1-CTF, restraining γ-secretase activity for Aβ production. Antagonist binding-induced conformational change of A2AR may attenuate the association with γ-secretase complex resulting in a “condensed” state of PS1, favoring Aβ generation.